Stepwise rotation of the γ‐subunit of EF0F1‐ATP synthase observed by intramolecular single‐molecule fluorescence resonance energy transfer 1

FEBS Letters

Reuter, Rolf¹  Börsch, Michael¹  Diez, Manuel¹  Gräber, Peter¹  Zimmermann, Boris¹ 

[1] Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 23 a, 79104 Freiburg, Germany

关键词: H+-ATP synthase;    EF0F1;    Inter-subunit rotation;    Single-molecule fluorescence resonance energy transfer;    FRET;    fluorescence resonance energy transfer;    AMPPNP;    adenosine-5′-(β;    γ-imido)triphosphate;    Cy5;    carbocyanine dye Cy5®;    TMR;    tetramethylrhodamine;   

DOI  :  10.1016/S0014-5793(02)03198-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The EF₀F₁-ATP synthase mutants bQ64C and γT106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b-subunit and with a cyanine (Cy5) at the γ-subunit. After reconstitution into liposomes, these double-labelled enzymes catalyzed ATP synthesis at a rate of 33 s⁻¹. Fluorescence of TMR and Cy5 was measured with a confocal set-up for single-molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with different fluorescence resonance energy transfer (FRET) efficiencies were observed. In the presence of ATP, repeating sequences of those three FRET-states were identified, indicating stepwise rotation of the γ-subunit of EF₀F₁.

【 授权许可】

Unknown